Porcine β-defensin 2 (PBD-2), expressed by different tissues of pigs, is a multifunctional cationic peptide with antimicrobial, immunomodulatory and growth-promoting abilities. As the latest generation of genome-editing tool, CRISPR/Cas9 system makes it possible to enhance the expression of PBD-2 in pigs by site-specific knock-in of pbd-2 gene into the pig genome. In this study, we aimed to generate marker-free pbd-2 knock-in pigs using the CRISPR/Cas9 and Cre/loxP systems. Two copies of pbd-2 gene linked by a T2A sequence were inserted into the porcine Rosa26 locus through CRISPR/Cas9-mediated homology-directed repair. The floxed selectable marker gene neoR , used for G418 screening of positive cell clones, was removed by cell-penetrating Cre recombinase with a recombination efficiency of 48.3%. Cloned piglets were produced via somatic cell nuclear transfer and correct insertion of pbd-2 genes was confirmed by PCR and Southern blot. Immunohistochemistry and immunofluorescence analyses indicated that expression levels of PBD-2 in different tissues of transgenic (TG) piglets were significantly higher than those of their wild-type (WT) littermates. Bactericidal assays demonstrated that there was a significant increase in the antimicrobial properties of the cell culture supernatants of porcine ear fibroblasts from the TG pigs in comparison to those from the WT pigs. Altogether, our study improved the protein expression level of PBD-2 in pigs by site-specific integration of pbd-2 into the pig genome, which not only provided an effective pig model to study the anti-infection mechanisms of PBD-2 but also a promising genetic material for the breeding of disease-resistant pigs.